In this renewal application I propose experiments to study how a neuroinvasive alpha herpesvirus (pseudorabies virus, PRV) infection drives axonal targeting of virions and viral proteins to promote anterograde spread of infection in the nervous system. These experiments take advantage of our technology advances in imaging, neuronal culturing systems, mass spectrometry, and deep sequencing, as well as new findings from the last funding period. In particular, we will focus on imaging US9, a critical protein for sorting virions into axons (aim 1). Using mass spectrometry and new enrichment techniques, we will determine the proteome of enriched vesicles containing GFP-US9 and gE-GFP and initiate studies to identify US9-specific protein complexes (aim 2). Finally, we will extend our work to test new hypotheses concerning the axonal damage response, the role of local axonal translation on viral axonal transport, and, using deep sequencing technology (Ilumina platform), how PRV infection affects not only axonal targeting of viral proteins, but also of host and viral mRNAs (aim 3). PUBLIC HEALTH RELEVANCE: The direction taken by the virus in a neuron after primary infection or reactivation from latency, as well as the extent of spread in a neuronal circuit can be the difference between a minor peripheral infection and lethal brain infection. My work will provide basic knowledge toward understanding how to block neuronal spread and its resulting damage. As viral tracing of neural circuitry has become an essential tool in the neuroscience community, our new discoveries will be immediately applicable for many ongoing fundamental research projects in neuroscience in a variety of animals with the promise to reveal detailed functional insights into neural circuit organization that have not been possible to achieve in the past.